Manuscript with Contributions from 636 All Co-Authors

1 Programmed cell death in diazotrophs and the fate of organic

2 matter in the western tropical South Pacific Ocean during the

3 OUTPACE cruise 4

5 Dina Spungin1, Natalia Belkin1, Rachel A. Foster2, Marcus Stenegren2, Andrea Caputo2, 6 Mireille Pujo-Pay3, Nathalie Leblond4, Cecile Dupouy4, Sophie Bonnet5, Ilana Berman- 7 Frank1*

8 9 1. The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel. 10 2. Stockholm University, Department of Ecology, Environment and Plant Sciences. Stockholm, Sweden. 11 3. Laboratoire d’Océanographie Microbienne – UMR 7321, CNRS - Sorbonne Universités, UPMC Univ Paris 06, 12 Observatoire Océanologique, 66650 Banyuls-sur-mer, France. 13 4. Observatoire Océanologique de Villefranche, Laboratoire d’Océanographie de Villefranche, UMR 7093, 14 Villefranche-surmer, France. 15 5. Aix-Marseille Univ., Univ. Toulon, CNRS/INSU, IRD, UM 110, Mediterranean Institute of Oceanography 16 (MIO) UM 110, 13288, Noumea, New Caledonia. 17 *Current address: Leon H. Charney School of Marine Sciences, University of Haifa, Mt. Carmel, 18 Haifa 3498838, Israel

19 20 Correspondence to: Ilana Berman-Frank ([email protected])

29 Abstract

30 The fate of diazotroph (N2 fixers) derived carbon (C) and nitrogen (N) and their contribution to vertical 31 export of C and N in the Western Tropical South Pacific Ocean was studied during the OUTPACE 32 experiment (Oligotrophy to UlTra-oligotrophy PACific Experiment). Our specific objective during 33 OUTPACE was to determine whether autocatalytic programmed cell death (PCD), occurring in some 34 diazotrophs, is an important mechanism affecting diazotroph mortality and a factor regulating the 35 vertical flux of organic matter and thus the fate of the blooms. We sampled at three long duration (LD) 36 stations of 5 days each (LDA, LDB, and LDC) where drifting sediment traps were deployed at 150, 325 37 and 500 m depth. LDA and LDB were characterized by high chlorophyll a (Chl a) concentrations (0.2- 38 0.6 µg L-1) and dominated by dense biomass of Trichodesmium as well as UCYN-B and diatom- 39 diazotroph associations (Rhizosolenia with Richelia-detected by microscopy and het-1 nifH copies). 40 Station LDC was located at an ultra-oligotrophic area of the South Pacific gyre with extremely low Chl 41 a concentration (~ 0.02 µg L-1) with limited biomass of diazotrophs predominantly the unicellular 42 UCYN-B. Our measurements of biomass from LDA and LDB yielded high activities of caspase-like 43 and metacaspase proteases that are indicative of PCD in Trichodesmium and other phytoplankton. 44 Metacaspase activity, reported here for the first time from oceanic populations, was highest at the surface 45 of both LDA and LDB, where we also obtained high concentrations of transparent exopolymeric 46 particles (TEP). TEP was negatively correlated with dissolved inorganic phosphorus and positively 47 coupled to both the dissolved and particulate organic carbon pools. Our results reflect the increase in 48 TEP production under nutrient stress and its role as a source of sticky carbon facilitating aggregation 49 and rapid vertical sinking. Evidence for bloom decline was observed at both LDA and LDB. However, 50 the physiological status and rates of decline of the blooms differed between the stations, influencing the 51 amount of accumulated diazotrophic organic matter and mass flux observed in the traps during our 52 experimental time frame. At LDA sediment traps contained the greatest export of particulate matter and 53 significant numbers of both intact and decaying Trichodesmium, UCYN-B, and het-1 compared to LDB 54 where the bloom decline began only 2 days prior to leaving the station and to LDC where no evidence 55 for bloom or bloom decline was seen. Substantiating previous findings from laboratory cultures linking 56 PCD to carbon export in Trichodesmium, our results from OUTPACE indicate that PCD may be induced 57 by nutrient limitation in high biomass blooms such as Trichodesmium or diatom-diazotroph associations. 58 Furthermore, PCD combined with high TEP production will tend to facilitate cellular aggregation and 59 bloom termination and will expedite vertical flux to depth. 60

61 62 63 64 65 66 67

68 1. Introduction

69 The efficiency of the biological pump, essential in the transfer and sequestration of carbon to the 70 deep ocean, depends on the balance between growth (production) and death. Moreover, the manner in 71 which marine organisms die may ultimately determine the flow of fixed organic matter within the 72 aquatic environment and whether organic matter is incorporated into higher trophic levels, recycled 73 within the microbial loop sustaining subsequent production, or sink out (and exported) to depth.

74 N2 fixing (diazotrophic) prokaryotic organisms are important contributors to the biological pump

75 and their ability to fix atmospheric N2 confers an inherent advantage in the nitrogen-limited surface 76 waters of many oceanic regions. The oligotrophic waters of the Western Tropical South Pacific

77 (WTSP) have been characterized with some of the highest recorded rates of N2 fixation (151-700 µmol 78 N m-2 d-1) (Garcia et al., 2007;Bonnet et al., 2005), and can reach up to 1200 µmol N m-2 d-1 (Bonnet et 79 al., 2017b). Diazotrophic communities comprised of unicellular cyanobacteria lineages (UCYN-A, B 80 and C), diatom-diazotroph associations such as Richelia associated with Rhizosolenia, and diverse

81 heterotrophic bacteria such as alpha and γ- protobacteria are responsible for these rates of N2 fixation. 82 The most conspicuous of all diazotrophs, and predominating in terms of biomass, is the filamentous 83 bloom-forming cyanobacteria Trichodesmium forming massive surface blooms that supply ~ 60-80 Tg -1 -1 84 N yr of the 100-200 Tg N yr of the estimated marine N2 fixation (Capone et al., 1997;Carpenter et 85 al., 2004;Westberry and Siegel,2006) with a large fraction fixed in the WTSP (Dupouy et al., 86 2000;Dupouy et al., 2011;Tenorio et al., in review) that may, based- on NanoSIMS cell-specific

87 measurements, contribute up to ~ 80 % of bulk N2 fixation rates in the WTSP (Bonnet et al., 2017a).

88 How Trichodesmium blooms form and develop has been investigated intensely while little data is 89 found regarding the fate of blooms. Trichodesmium blooms often collapse within 3-5 days, with 90 mortality rates paralleling bloom development rates (Rodier and Le Borgne, 2008;Rodier and Le 91 Borgne, 2010;Bergman et al., 2012). Cell mortality can occur due to grazing (O'Neil, 1998), viral lysis 92 (Hewson et al., 2004;Ohki, 1999), and/or programmed cell death (PCD) an autocatalytic genetically 93 controlled death (Berman-Frank et al., 2004). PCD is induced in response to oxidative and nutrient 94 stress, as has been documented in both laboratory and natural populations of Trichodesmium (Berman- 95 Frank et al., 2004;Berman-Frank et al., 2007) and in other phytoplankton (Bidle, 2015). The cellular 96 and morphological features of PCD in Trichodesmium, include elevated gene expression and activity 97 of metacaspases and caspase like-proteins; hallmark protein families involved in PCD pathways in 98 other organisms whose functions in Trichodesmium are currently unknown. PCD in Trichodesmium 99 also displays increased production of transparent exopolymeric particles (TEP) and trichome 100 aggregation as well as buoyancy loss via reduction in gas vesicles. This causes rapid sinking rates that 101 can be significant when large biomass such as that in oceanic blooms crashes (Bar-Zeev et al., 102 2013;Berman-Frank et al., 2004).

103 Simulating PCD in laboratory cultures of Trichodesmium in 2 m water columns (Bar-Zeev et al., 104 2013) led to a collapse of the Trichodesmium biomass and to greatly enhanced sinking of large 105 aggregates reaching rates of up to ~ 200 m d-1 that efficiently exported particulate organic carbon 106 (POC) and particulate organic nitrogen (PON) to the bottom of the water column. Although the 107 sinking rates and degree of export from this model system could not be extrapolated to the ocean, this 108 study mechanistically linked autocatalytic PCD and bloom collapse to quantitative C and N export 109 fluxes, suggesting that PCD may have an impact on the biological pump efficiency in the oceans (Bar- 110 Zeev et al., 2013).

111 We further examined this issue in the open ocean and investigated the cellular processes mediating 112 Trichodesmium mortality in a large surface bloom from the New Caledonian lagoon (Spungin et al., 113 2016). Nutrient stress induced a PCD mediated crash of the Trichodesmium bloom. The filaments and 114 colonies were characterized by upregulated expression of metacaspase genes, downregulated 115 expression of gas-vesicle genes, enhanced TEP production, and aggregation of the biomass (Spungin 116 et al., 2016). Due to experimental conditions we could not measure the subsequent export and vertical 117 flux of the dying biomass in the open ocean. Moreover, while the existence and role of PCD and its 118 mediation of biogeochemical cycling of organic matter has been investigated in Trichodesmium, 119 scarce information exists about PCD and other mortality pathways of most marine diazotrophs.

120 The OUTPACE (Oligotrophy to UlTra-oligotrophy PACific Experiment) cruise was conducted 121 from 18 February to 3 April 2015 along a west to east gradient from the oligotrophic area north of 122 New Caledonia to the ultraoligotrophic western South Pacific gyre (French Polynesia). The goal of the 123 OUTPACE experiment was to study the diazotrophic blooms and their fate within the oligotrophic 124 ocean in the Western Tropical South Pacific Ocean (Moutin et al., 2017). Our specific objective was to 125 determine whether PCD was an important mechanism affecting diazotroph mortality and a factor 126 regulating the fate of the blooms by mediation of vertical flux of organic matter. The strategy and 127 experimental approach of the OUTPACE transect enabled sampling at three long duration (LD) 128 stations of 5 days each (referred to as stations LDA, LDB, and LDC) and provided 5-day snapshots 129 into diazotroph physiology, dynamics, and mortality processes. We specifically probed for the 130 induction and operation of PCD and examined the relationship of PCD to the fate of organic matter 131 and vertical flux from diazotrophs by the deployment of 3 sediment traps at 150, 325 and 500 m 132 depths.

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137 2. Methods 138 139 2.1. Sampling site and sampling conditions 140 Sampling was conducted on a transect during austral summer (18 Feb-5 Apr, 2015), on board the 141 R/V L’Atalante (Moutin et al., 2017). Samples were collected from three long duration stations (LD- 142 A, LD-B and LD-C) where the ship remained for 5 days at each location and 15 short duration (SD1- 143 15) stations (approximately eight hours duration). The cruise transect was divided into two geographic 144 regions. The first region (Melanesian archipelago, MA) included SD1-12, LDA and LDB stations 145 (160º E-178º E and 170º-175º W). The second region (subtropical gyre, GY) included SD 13-15 and 146 LDC stations (160º W-169º W). 147 148 2.2. Chlorophyll a 149 Samples for determination of (Chl a) concentrations were collected by filtering 550 ml sea water 150 on GF/F filters (Whatman, UK). Filters were frozen and stored in liquid nitrogen, Chl a was extracted 151 in methanol and measured fluorometrically (Turner Designs Trilogy Optical kit) (Le Bouteiller et al., 152 1992). Satellite derived surface Chl a concentrations at the LD stations were used from before and 153 after the cruise sampling at the LD stations. Satellite Chl a data are added as supplementary video files 154 (Supplementary videos S1, S2, S3). 155 156 2.3. Caspase and metacaspase activities

157 Biomass was collected on 25 mm, 0.2 µm pore-size polycarbonate filters and resuspended in 0.6-1 158 ml Lauber buffer [50 mM HEPES (pH 7.3), 100 mM NaCl, 10 % sucrose, 0.1 % (3-cholamidopropyl)- 159 dimethylammonio-1-propanesulfonate, and 10 mM dithiothreitol] and sonicated on ice (four cycles of 160 30 seconds each) using an ultracell disruptor (Sonic Dismembrator, Fisher Scientific, Waltham, MA, 161 USA). Cell extracts were centrifuged (10,000 x g, 2 min, room temperature), and the supernatant was 162 collected for caspase and metacaspase activity measurements. Caspase specific activity (normalized to 163 total protein concentration) was determined by measuring the kinetics of cleavage for the fluorogenic 164 caspase substrate Z-IETD-AFC (Z-Ile-Glu-Thr-Asp-AFC) at a 50 mM final concentration (using Ex 165 400 nm, Em 505 nm; Synergy4 BioTek, Winooski, VT, USA), as previously described in Bar-Zeev et 166 al. (2013). Metacaspase specific activity (normalized to total protein concentration) was determined by 167 measuring the kinetics of cleavage for the fluorogenic metacaspase substrate Ac-VRPR-AMC (Ac- 168 Val-Arg-Pro-Arg-AMC), (Klemenčič et al., 2015;Tsiatsiani et al., 2011) at a 50 mM final 169 concentration (using Ex 380 nm, Em 460 nm; Synergy4 BioTek, Winooski, VT, USA) (Klemenčič et 170 al., 2015;Tsiatsiani et al., 2011). Relative fluorescence units were converted to protein-normalized 171 substrate cleavage rates using AFC and AMC standards (Sigma) for caspase and metacaspase 172 activities, respectively. Total protein concentrations were determined by Pierce™ BCA protein assay 173 kit (Thermo Scientific product #23225).

174 2.4. Phosphate analysis 3- 175 Seawater for phosphate (PO4 , DIP) analysis was collected in 20 mL high-density polyethylene −1 176 HCL-rinsed bottles and poisoned with HgCl2 to a final concentration of 20 μg L , stored at 4 °C until 3- 177 analysis. PO4 was determined by a standard colorimetric technique using a segmented flow analyzer 178 according to Aminot and Kérouel (2007) on a SEAL Analytical AA3 HR system 20 (SEAL Analytica, 3- 179 Serblabo Technologies, Entraigues Sur La Sorgue, France). Quantification limit for PO4 was 0.05 180 µmol L-1. 181 182 2.5. Particulate organic carbon (POC) and nitrogen (PON)

183 Samples were filtered through pre-combusted (4 h, 450 ºC) GF/F filters (Whatman GF/F, 25 mm), 184 dried overnight at 60 ºC and stored in a desiccator until further analysis. POC and PON were 185 determined using a CHN analyzer Perkin Elmer (Waltham, MA, USA) 2400 Series II CHNS/O 186 Elemental Analyzer after carbonate removal from the filters using overnight fuming with concentrated 187 HCl vapor. 188 189 2.6. Dissolved organic carbon (DOC) and Total organic carbon (TOC)

190 Samples were collected from the Niskin bottles in combusted glass bottles and were immediately 191 filtered through 2 precombusted (24 h, 450 ºC) glass fiber filters (Whatman GF/F, 25 mm). Filtered 192 samples were collected into glass precombusted ampoules that where sealed immediately after

193 filtration. Samples were acidified with orthophosphoric acid (H3PO4) and analyzed by high 194 temperature catalytic oxidation (HTCO) (Sugimura and Suzuki, 1988;Cauwet, 1994) on a Shimadzu 195 TOC-L analyzer. TOC was determined as POC+DOC.

196

197 2.7. Transparent exopolymeric particles (TEP) 198 Water samples (100 mL) were gently (< 150 mbar) filtered through a 0.45 µm polycarbonate filter 199 (GE Water & Process Technologies). Filters were then stained with a solution of 0.02 % Alcian Blue 200 (AB) and 0.06 % acetic acid (pH of 2.5), and the excess dye was removed by a quick deionized water 201 rinse. Filters were then immersed in sulfuric acid (80 %) for 2 h, and the absorbance (787 nm) was 202 measured spectrophotometrically (CARY 100, Varian). AB was calibrated using a purified 203 polysaccharide gum xanthan (GX) (Passow and Alldredge, 1995). TEP concentrations (µg GX 204 equivalents L-1) were measured according to Passow and Alldredge (1995). To estimate the role of 205 TEP in C cycling, the total amount of TEP-C was calculated using the TEP concentrations at each 206 depth, and the conversion of GX equivalents to carbon applying the revised factor of 0.63 based on 207 empirical experiments from both natural samples from different oceanic areas and phytoplankton 208 cultures (Engel, 2004).

209 2.8. Diazotrophic abundance

210 The full description of DNA extraction, primer design and qPCR analyses are described in detail in 211 this issue (Stenegren et al., 2017). Briefly, 2.5 L of water from 6-7 depths with surface irradiance light 212 intensity (100, 75, 54, 36, 10, 1, and 0.1 %) were sampled and filtered onto a 25 mm diameter Supor 213 filter (Pall Corporation, PallNorden, AB Lund Sweden) with a pore size 0.2 µm filters. Filters were 214 stored frozen in pre-sterilized bead beater tubes (Biospec Bartlesville Ok, USA) containing 30 mL of 215 0.1 mm and 0.5 mm glass bead mixture. DNA was extracted from the filters using a modified protocol 216 of the Qiagen DNAeasy plant kit (Moisander et al., 2008) and eluted in 70 µL. With the re-eluted DNA 217 extracts ready, samples were analyzed using the qPCR instrument StepOnePlus (Applied Biosystems) 218 and fast mode. Previously designed TaqMAN assays and oligonucleotides and standards were prepared 219 in advance and followed previously described methods for the following cyanobacterial diazotrophs: 220 Trichodesmium, UCYN-A1, UCYN-A2, UCYN-B, Richelia symbionts of diatoms (het-1, het-2, het-3) 221 (Stenegren et al., 2017;Church et al., 2005;Foster et al., 2007;Moisander et al., 2010;Thompson et al., 222 2012). .

223 2.9. Microscopy

224 Samples for microscopy were collected in parallel from the same depth profiles for nucleic acid as 225 described in Stenegren et al. (2017). Briefly, 2 profiles were collected on day 1 and 3 at each LD station 226 and immediately filtered onto a 47 mm diameter Poretics (Millipore, Merck Millipore, Solna, 227 Sweden) membrane filter with a pore size of 5 µm using a peristaltic pump. After filtration samples 228 were fixed with a 1 % paraformaldehyde (v/v) for 30 min. prior to storing at -20 °C. The filters were 229 later mounted onto an oversized slide and examined under an Olympus BX60 microscope equipped with 230 blue (460-490 nm) and green (545-580 nm) excitation wavelengths. Three areas (0.94 mm2) per filter 231 were counted separately and values were averaged. When abundances were low, the entire filter 232 (area=1734 mm2) was observed and cells enumerated. Due to poor fluorescence, only Trichodesmium 233 colonies and free-filaments could be accurately enumerated by microscopy, and in addition the larger 234 cell diameter Trichodesmium (Katagynemene pelagicum) was counted separately as these were often 235 present (albeit at lower densities). Other cyanobacterial diazotrophs (e.g. Crocosphaera watsonii-like 236 cells, the Richelia symbionts of diatoms were present but with poor fluorescence and could only be 237 qualitatively noted.

238 2.10. Particulate matter from sediment traps

239 Particulate matter export was quantified with three PPS5 sediment traps (1 m2 surface collection, 240 Technicap, France) deployed for 5 days at 150, 325 and 500 m at each LD station. Particle export was 241 recovered in polyethylene flasks screwed on a rotary disk which allowed flasks to be changed 242 automatically every 24-h to obtain a daily material recovery. The flasks were previously filled with a

243 buffered solution of formaldehyde (final conc. 2 %) and were stored at 4 °C until analysis to prevent 244 degradation of the collected material. The flask corresponding to the fifth day of sampling on the 245 rotary disk was not filled with formaldehyde to collect ‘fresh particulate matter’ for further diazotroph 246 quantification. Exported particulate matter was weighed and analyzed on EA-IRMS (Integra2, Sercon 247 Ltd) to quantify exported PC and PN.

248 2.11. Diazotroph abundance in the traps

249 Triplicate aliquots of 2-4 mL from the flask dedicated for diazotroph quantification were filtered 250 onto 0.2 µm Supor filters, flash frozen in liquid nitrogen and stored at -80 °C until analysis. Nucleic 251 acids were extracted from the filters as described in Moisander et al. (2008) with a 30 second 252 reduction in the agitation step in a Fast Prep cell disruptor (Thermo, Model FP120; Qbiogene, Inc. 253 Cedex, Frame) and an elution volume of 70 µl. Diazotroph abundance for Trichodesmium spp., 254 UCYN-B, UCYN-A1, het-1, and het-2 were quantified by qPCR analyses on the nifH gene using 255 previously described oligonucleotides and assays (Foster et al., 2007;Church et al., 2005). The qPCR 256 was conducted using a StepOnePlus system (applied Biosystems, Life Technologies, Stockholm 257 Sweden) with the following parameters: 50 °C for 2 min, 95°C for 10 min, and 45 cycles of 95°C for 258 15s followed by 60°C for 1 min. Gene copy numbers were calculated from the mean cycle threshold 259 (Ct) value of three replicates and the standard curve for the appropriate primer and probe set. For each 260 primer and probe set, duplicate standard curves were made from 10-fold dilution series ranging from 261 108 to 1 gene copies per reaction. The standard curves were made from linearized plasmids of the 262 target nifH or from synthesized gBLocks gene fragments (IDT technologies, Cralville, Iowa USA). 263 Regression analyses of the results (number of cycles=Ct) of the standard curves were analyzed in 264 Excel. 2 µl of 5 KDa filtered nuclease free water was used for the no template controls (NTCs). No 265 nifH copies were detected for any target in the NTC. In some samples only 1 or 2 of the 3 replicates 266 produced an amplification signal; these were noted as detectable but not quantifiable (dnq). A 4th 267 replicate was used to estimate the reaction efficiency for the Trichodesmium and UCYN-B targets as 268 previously described in (Short et al., 2004). Seven and two samples were below 95 % in reaction 269 efficiency for Trichodesmium and UCYN-B, respectively. The detection limit for the qPCR assays is 270 1-10 copies.

271 2.12. Statistics 272 A Spearman correlation coefficient test was applied to examine the strength of association 273 between two variables and the direction of the relationship. 274

275 3. Results and discussion 276 3.1. Diazotrophic characteristics and abundance in the LD stations

277 The sampling strategy of the transect was planned so that changes in abundance and fate of 278 diazotrophs could be followed in “long duration” (LD) stations where measurements were taken from 279 the same water mass (and location) over 5 days and drifting sediment traps were deployed (Moutin et 280 al., 2017). Although rates for the different parameters were obtained for 5 days, this period is still a 281 “snapshot” in time with the processes measured influenced by preceding events also continuing after 282 the ship departed. Specifically, production of photosynthetic biomass (as determined from satellite- 283 derived Chl a) and development of surface phytoplankton blooms, including cyanobacterial 284 diazotrophs, displayed specific characteristics for each of the LD stations. We first examined the 285 satellite-derived surface Chl a concentrations by looking at changes around the LD stations before and 286 after our 5-day sampling at each station [daily surface Chl a (mg m-3)] (Supplementary videos S1, S2, 287 S3).

288 At LDA, satellite data confirmed high concentrations of Chl a indicative of intense surface blooms 289 (~ 0.55 µg L-1) between 8th of February 2015 to 19th of February 2015 which began to gradually 290 decline with over 60 % Chl a reduction until day 1 at the station (Supplementary video S1, Fig. 1a). 291 By the time we reached LDA on 25.02.15 (day 1) Chl a concentrations averaged ~ 0.2 µg L-1 Chl a at 292 the surface (Fig. 1a) and remained steady for the next 5 days with Chl a values of 0.2 µg L-1 measured 293 on day 5 (Fig 1a). When looking for biomass at depth the DCM recorded at ~ 80 m depth was 294 characterized by Chl a concentrations increasing from 0.4 to 0.5 µg L-1 between day 3 and 5 295 respectively (Fig. 1d). While the Chl a values of the surface biomass decreased for approximately one 296 week prior to our sampling at station, the Chl a concentrations measured at depth increased during the 297 corresponding time.

298 In contrast to LDA, the satellite data from LDB confirmed the presence of a surface bloom/s for 299 over one month prior to our arrival at the station on 15th of March 2015 (day 1) (Supplementary video 300 S2, Fig. 1b). This bloom was characterized by high surface Chl a concentrations (~ 0.6 µg L-1, 301 Supplementary video S2) and on day 1 at the station surface Chl a was 0.6 µg L-1 (Fig. 1b). Surface 302 Chl a then decreased over the next days at the station with a 50 % reduction of Chl a concentration 303 from the sea surface (5m) on day 5 (0.4 µg L-1), (Fig. 1e). Thus, it appears that our 5 sampling days at 304 LDB were tracking a surface bloom that had only began to decline after day 3 and continued to 305 decrease (~ 0.1 µg L-1) also after we have left (Fig.1b). On day 1 of sampling, the DCM at LDB was 306 relatively shallow, at 40 m with Chl a values of 0.5 µg L-1. By day 5 the DCM had deepened to 80 m 307 (de Verneil et al., 2017).

308 LDC was located in a region of extreme oligotrophy within the Cook Islands territorial waters 309 (GY waters). This station was characterized historically (~ 4 weeks before arrival) by extremely low

310 Chl a concentrations at the surface (~ 0.02 µg L-1, Supplementary video S3) that were an order of 311 magnitude lower than average Chl a measured at LDA and LDB. These values remained low with no 312 significant variability for the 5 days at station or later (Fig. 1f) (Supplementary video S3, Fig. 1c). 313 Similar to the results from LDA, the DCM at LDC was found near the bottom of the photic layer at ~ 314 135 m, with Chl a concentrations about 10-fold higher than those measured at surface with ~ 0.2 µg 315 L-1 (Fig. 1f).

316 Chl a is an indirect proxy of photosynthetic biomass and we thus needed to ascertain who the 317 dominant players (specifically targeting diazotrophic populations) were at each of the LD stations. 318 Moreover, At LDA and LDB diazotrophic composition and abundance as determined by qPCR 319 analysis were quite similar. At LDA Trichodesmium was the most abundant diazotroph, ranging 320 between 6x104 -1x106 nifH copies L-1 in the upper water column (0-70 m). UCYN-B (genetically 321 identical to Crocosphaera watsonii) co-occurred with Trichodesmium between 35 and 70 m, and het1 322 specifically identifying the diatom-diazotroph association (DDA) between the diatom Rhizosolenia 323 and the heterocystous diazotroph Richelia, was observed only at the surface waters at 4 m. UCYN-B 324 and het-1 abundances were relatively lower than Trichodesmium abundances with 2x102 nifH copies 325 L-1 and 3x103 nifH copies L-1 respectively (Stenegren et al., 2017). Microscopic observations from 326 LDA indicated that near the surface Rhizosolenia populations were already showing signs of decay 327 since the silicified cell-wall frustules were broken and free filaments of Richelia were observed (Fig.

328 2e-f) (Stenegren et al., 2017). DDAs are significant N2 fixers in the oligotrophic oceans. Although 329 their abundance in the WTSP is usually low, they are common and highly abundant in the New 330 Caledonian lagoon significantly impacting C sequestration and rapid sinking (Turk-Kubo et al., 2015).

331 At LDB, Trichodesmium was also the most abundant diazotroph with nifH copies L-1 ranging 332 between 1x104-5x105 within the top 60 m (Stenegren et al., 2017). Microscopical analyses confirmed 333 high abundance of free filaments of Trichodesmium at LDB, while colonies were rarely observed 334 (Stenegren et al., 2017). Observations of poor cell integrity were reported for most collected samples, 335 with filaments at various stages of degradation and colonies under possible stress (Fig. 2a-d). In 336 addition to Trichodesmium, UCYN-B was the second most abundant diazotroph ranging between 337 1x102 and 2x103 nifH copies L-1. Other unicellular diazotrophs of the UCYN groups (UCYN-A1 and 338 UCYN-A2) were the least detected diazotrophs (Stenegren et al., 2017). Of the three heterocystous 339 cyanobacterial symbiont lineages (het-1, het-2, het-3), het-1 was the most dominant (1x101-4x103 nifH 340 copies L-1), (Stenegren et al., 2017). Microscopic analyses from LDB demonstrated the co-occurrence 341 of degrading diatom cells, mainly belonging to Rhizosolenia (Stenegren et al., 2017) (Fig. 2e-f).

342 In contrast to LDA and LDB, at LDC, the highest nifH copy numbers (up to 6x105 nifH copies L-1 at 343 60 m depth were from the unicellular diazotrophs UCYN-B (Stenegren et al., 2017) Trichodesmium

344 was only detected at 60 m and with very low copy numbers of nifH (~7x102 nifH copies L-1) 345 (Stenegren et al., 2017).

-1 346 Corresponding to the physiological status of the bloom, higher N2 fixation rates (45.0 nmol N L 347 d-1) were measured in the surface waters (5m) of LDB in comparison with those measured at LDA and 348 LDC (19.3 nmol N L-1 d -1 in LDA and below the detection limit at LDC at 5m), (Caffin et al., 2017). 349 350 3.2. Diazotrophic bloom demise in the LD stations 351 Of the 3 long duration stations we examined, LDA and LDB had a higher biomass of diazotrophs 352 during the 5 days of sampling (section 3.1). Our analyses examining bloom dynamics from the 353 satellite-derived Chl a concentrations indicate a declining trend in chlorophyll-based biomass during 354 the sampling time period. Yet, both LDA and LDB were still characterized by high (and visible to the 355 eye at surface) biomass on the first sampling day at each station (day 1) as determined by qPCR and 356 microscopy (Stenegren et al., 2017). This is different from LDC where biomass was extremely limited, 357 and no clear evidence was obtained for any specific bloom or bloom demise. We therefore show 358 results mostly from LDA and LDB and focus specifically on the evidence for PCD and diazotroph 359 decline in areas with high biomass and surface blooms.

360 The mortality of phytoplankton at sea can be difficult to discern as it most probably results 361 from co-occurring processes including physical forces, chemical stressors, grazing, viral lysis, and/or 362 PCD. Here, we specifically focused on evidence for PCD and whether the influence of zooplankton 363 grazing on the diazotrophs and especially on Trichodesmium at LDA and LDB impacted bloom 364 dynamics. At LDA and LDB total zooplankton population was generally low. Total zooplankton 365 population at LDA ranged between 911-1900 individuals m-3 and in LDB between 1209-2188 366 individuals m-3 on day 1 and day 5 respectively. Trichodesmium is toxic and inedible to most 367 zooplankton excluding three species of harpacticoid zooplankton- Macrosettella gracilis, Miracia 368 efferata and Oculosetella gracilis (O'Neil and Roman, 1994). During our sampling days at these 369 stations, Macrosettella gracilis a specific grazer of Trichodesmium comprised less than 1 % of the 370 total zooplankton community with another grazer Miracia efferata comprising less than 0.1 % of total 371 zooplankton community. Oculosetella gracilis was not found at these stations. The low number of 372 harpacticoid zooplankton specifically grazing on Trichodesmium found in the LDA and LDB station, 373 refutes the possibility that grazing caused the massive demise of the bloom. Moreover, the toxicity of 374 Trichodesmium to many grazers (Rodier and Le Borgne, 2008;Kerbrat et al., 2011) could critically 375 limit the amount of Trichodesmium-derived recycled matter within the upper mixed layer.

376 Viruses have been increasingly invoked as key agents terminating phytoplankton blooms. 377 Phages may infect Trichodesmium (Brown et al., 2013; Hewson et al., 2004; Ohki, 1999) yet they 378 have not been demonstrated to terminate large surface blooms. Virus like particles were previously 379 enumerated from Trichodesmium samples during bloom demise, yet the numbers of virus-like

380 particles did not indicate that a massive, phage-induced lytic event of Trichodesmium occurred there 381 (Spungin et al., 2016). Virus infection may cause for the induction of PCD by causing an increased 382 production of reactive oxygen species (Vardi et al., 2012) which stimulates PCD in algal cells 383 (Berman-Frank et al., 2004; Bidle, 2015; Thamatrakoln et al., 2012). Viral attack can also directly 384 trigger PCD as part of an antiviral defense system (Bidle, 2015). Virus abundance and activity were 385 not enumerated in this study, so we cannot estimate their specific influence on mortality.

386 Limited availability of Fe and P induce PCD in Trichodesmium (Berman-Frank et al. 2004; 387 Bar-Zeev et al. 2013). At LDA and LDB, Fe concentrations at the time of sampling were relatively 388 high (> 0.5 nM), possibly due to island effects (de Verneil et al., 2017). Phosphorus availability, or 3- 389 lack of phosphorus, can also induce PCD (Berman-Frank et al., 2004;Spungin et al., 2016). PO4 390 concentrations at the surface (0-40m) of LDA and LDB stations were extremely low around 0.05 µmol 391 L-1 (de Verneil et al., 2017), possibly consumed by the high biomass and high growth rates of the 3- 392 bloom causing nutrient stress and bloom mortality. PO4 concentrations observed at LDC were above 393 the quantification limit with average values of 0.2 µmol L-1 in the 0-150 m depths (data not shown). 394 These limited P concentrations may curtail the extent of growth, induce PCD, and pose an upper limit 395 on biomass accumulation.

396 Here we compared, for the first time in oceanic populations, two PCD indices, caspase-like 397 and metacaspase activities, to examine the presence/operation of PCD in the predominant 398 phytoplankton (and diazotroph) populations along the transect. This was determined by the cleavage 399 of Z-IETD-AFC and Ac-VRPR-AFC substrates for caspase-like and metacaspase activities 400 respectively. We specifically show the results from LDA and LDB where biomass and activities were 401 detectable.

402 Classic caspases are absent in phytoplankton, including in cyanobacteria, and are unique to 403 metazoans and several viruses (Minina et al., 2017). In diverse phytoplankton the presence of a 404 caspase domain suffices to demonstrate caspase-like proteolytic activity that occurs upon PCD 405 induction when the caspase specific substrate Z-IETD-AFC is added (Berman-Frank et al., 2004;Bidle 406 and Bender, 2008;Bar-Zeev et al., 2013). Cyanobacteria and many diazotrophs contain genes that are 407 similar to caspases, the metacaspases-cysteine proteases. These proteases share structural properties 408 with caspases, specifically a histidine-cysteine catalytic dyad in the predicted active site (Tsiatsiani et 409 al., 2011). While the specific role and function/s of metacaspases genes are unknown, and cannot be 410 directly linked to gene expression, preliminary investigations have indicated that when PCD is induced 411 some of these genes are upregulated (Bidle and Bender, 2008;Spungin et al., 2016).

412 Of the abundant diazotrophic populations at LDA and LDB 12 metacaspases have previously 413 been identified in Trichodesmium spp. (Asplund-Samuelsson et al., 2012;Asplund‐Samuelsson, 414 2015;Jiang et al., 2010;Spungin et al., 2016). Phylogenetic analysis of a wide diversity of truncated

415 metacaspase proteins, containing the conserved and characteristic caspase super family (CASc; 416 cl00042) domain structure, revealed metacaspase genes in both Richelia intracellularis (het-1) from 417 the diatom-diazotroph association and Crocosphaera watsonii (a cultivated unicellular 418 cyanobacterium) which is genetically identical to the UCYN-B nifH sequences (Spungin et al., 419 unpublished data).

420 We compared between metacaspase and caspase-like activities for the > 0.2 µm fraction 421 sampled assuming that the greatest activity would be due to the principle organisms contributing to the 422 biomass – i.e. the diazotrophic cyanobacteria. Caspase-like activity and metacaspase activity were 423 specifically measured at all LD stations (days 1,3,5) at 5 depths between 0-200 m. Caspase-like 424 activity at the surface waters (50 m) at LDA, as determined by the cleavage of IETD-AFC substrate, 425 was between 2.3 to 2.8±0.1 pM hydrolyzed mg protein-1 min-1 on days 1 and 3 respectively (Fig. 3a). 426 The highest activity was measured on day 5 at 50 m with 5.1±0.1 pM hydrolyzed mg protein-1 min-1. 427 Similar trends were obtained at LDA for metacaspase activity as measured by the cleavage of the 428 VRPR-AMC substrate, containing an Arg residue at the P1 position, specific for metacaspase 429 cleavage, (Tsiatsiani et al., 2011;Klemenčič et al., 2015). High and similar metacaspase activities were 430 measured on days 1 and 3 (50 m) with 32±4 and-35±0.2 pM hydrolyzed mg protein-1 min-1 431 respectively (Fig. 3a). The highest metacaspase activity was measured on day 5 at 50 m with 59±1 pM 432 hydrolyzed mg protein-1 min-1 with declining activity at greater depths (Fig. 3b).

433 Caspase-like activity at LDB, was similar for all sampling days, with the highest activity 434 recorded from the surface samples (ranging from 3±0.1 to 4.5±0.2 pM hydrolyzed mg protein-1 min-1 435 at 7 m depth and then decreasing with depth) (Fig. 3d). At day 3 caspase-like activity at LDB 436 increased at the surface with 4.5±0.2 pM hydrolyzed mg protein-1 min-1 and then declined slightly by 437 day 5 back to 3±0.1 pM hydrolyzed mg protein-1 min-1. The decrease in activity at the surface between 438 day 3 and 5 was accompanied by an increase in caspase-like activity measured in the DCM between 439 day 3 and 5 (Fig. 3d). Caspase-like activity at the DCM at day 3 (35 m) was 1±0.4 pM hydrolyzed mg 440 protein-1 min-1 and by day 5 increased to 3±0.1 pM hydrolyzed mg protein-1 min-1 for samples from 70 441 m depth. Thus, at LDB, caspase-like activity increased from day 1 to 5 and with depth, with higher 442 activities that initially were recorded at surface and then at depth coupled with the decline of the 443 bloom (Fig. 3d). Similar trends were obtained at LDB for metacaspase activity with 11.1±0.9 pM 444 hydrolyzed mg protein-1 min-1 at the surface (7 m) on day 1. A 4-fold increase in activity was 445 measured at the surface on day 3 with 40.1±5 pM hydrolyzed mg protein-1 min-1 (Fig. 3e). Similar high 446 activities were measured also on day 5 (Fig. 3e). However, the increase in activity was also 447 pronounced at depth of ~ 70 m and not only at the surface. Metacaspase activity at day 5 was the 448 highest with 40.3±0.5 and 44.6±5 pM hydrolyzed mg protein-1 min-1 at 7 and 70 m respectively (Fig 449 3e). The relatively low metacaspase activity measured on day 1 appears to correspond with the

450 stressed physiological status of the biomass just prior to increased mortality rates. Metacaspase 451 activity increased corresponding with the pronounced decline in Chl a from day 1 to day 5 (Fig. 1b).

452 The measured metacaspase activities were typically 10-fold higher than caspase-like activity 453 rates (Fig 3). Yet, metacaspase and caspase-like activities are significantly and positively correlated at 454 LDA and LDB (r=0.8, p<0.05 and r=0.8 p<0.001 for LDA and LDB respectively) (Fig. 3c and 3f). 455 Both findings (i.e. higher metacaspase activity and tight correlation between metacaspase and 456 caspases) were demonstrated specifically in cultures and natural populations of Trichodesmium 457 undergoing PCD (Spungin et al., in review). As our experiments find a significant positive correlation 458 between both activates, we have done a series of inhibitor experiments to test whether metacaspase are 459 substrate specific and are not the caspase-like activity we have examined (Spungin et al., in review). In 460 vitro treatment with a metacaspase inhibitor- antipain dihydrochloride, efficiently inhibited 461 metacaspase activity, confirming the arginine-based specificity of Trichodesmium. Our biochemical 462 activity and inhibitor observations demonstrate that metacaspases and caspases-like activities are 463 likely distinct and are independently activated under stress and coupled to PCD in our experiments of 464 both laboratory and field populations. However, caspase-like activity was somewhat sensitive to the 465 metacaspase inhibitor, antipain, showing a ~30-40% drop in activity. This hints at some catalytic 466 crossover between these two catalytic activities in Trichodesmium that further should be studied. We 467 do not know what protein is responsible for the caspase-specific activities and what drivers regulate it. 468 Yet, the tight correlation between both activities specifically for Trichodesmium, and here at LDA and 469 LDB suggest that both activities occur in the cell when PCD is induced. To date, we are not aware of 470 any previous studies examining metacaspase or caspase-like activity (or the existence of PCD) in 471 diatom-diazotroph associations such as Rhizolsolenia-Richelia.

472 3.3. TEP dynamics and carbon pools

473 Transparent exopoloymeric particles link between the particulate and dissolved carbon fractions 474 and act to augment the coagulation of colloidal precursors from the dissolved organic matter and from 475 biotic debris and to increase vertical carbon flux (Passow, 2002;Verdugo and Santschi, 2010). TEP 476 production also increases upon PCD induction – specifically in large bloom forming organisms such 477 as Trichodesmium (Berman-Frank et al., 2007;Bar-Zeev et al., 2013).

478 At LDA, TEP concentrations at 50 m depth were highest at day 1 with measured concentrations 479 of 562±7 µg GX L-1 (Table. 1) that appear to correspond with the declining physiological status of the 480 cells that were sampled at that time (Fig. 2a-d). TEP concentrations during days 3 and 5 decreased to 481 less than 350 µg GX L-1, and it is possible that most of the TEP had been formed and sank prior to our 482 measurements in the LDA station.

483 At LDB, TEP concentrations at day 1 and 3 were similar with ~ 400 µg GX L-1 at the surface (7 484 m) while concentrations decreased about 2-fold with depth, averaging at 220±56 and 253±32 µg GX

485 L-1 (35-200 m) for day 1 and 3 respectively (Fig. 4a, Table 2). A significant (> 150 %) increase in TEP 486 concentrations was observed on day 5 compared to previous days, with TEP values of 597±69 µg GX 487 L-1 at the surface (7m) (Fig 4b, Table 2). Although TEP concentrations were elevated at surface, the 488 difference in averaged TEP concentrations observed at the deeper depths (35-200 m) between day 3 489 (157±28 µg GX L-1) and day 5 (253±32 GX L-1) indicated that TEP from the surface was either 490 breaking down or sinking to depth (Fig. 4a, Table 2). The TEP concentrations from this study 491 correspond with values and trends reported from other marine environments (Engel, 2004;Bar-Zeev et 492 al., 2009) and specifically with TEP concentrations measured from the New Caledonian lagoon 493 (Berman-Frank et al., 2016).

494 TEP is produced by many phytoplankton including cyanobacteria under conditions uncoupling 495 growth from photosynthesis (i.e. nutrient but not carbon limitation) (Berman-Frank and Dubinsky, 496 1999;Passow, 2002;Berman-Frank et al., 2007). Decreasing availability of dissolved nutrients such as 497 nitrate and phosphate has been significantly correlated with increase in TEP concentrations in both 498 cultured phytoplankton and natural marine systems (Bar-Zeev et al., 2013;Brussaard et al., 2005;Engel 499 et al., 2002;Urbani et al., 2005). TEP production in Trichodesmium is enhanced as a function of 500 nutrient stress (Berman-Frank et al., 2007).

501 In the New Caledonian coral lagoon TEP concentrations were significantly and negatively 502 correlated with ambient concentrations of dissolved inorganic phosphorus (DIP) (Berman-Frank et al., 503 2016). Here, at LDB a significant negative correlation of TEP with DIP was also observed (Fig. 4b, 504 p=0.005), suggesting that lack of phosphorus set a limit to continued biomass increase and stimulated 505 TEP production in the nutrient-stressed cells. TEP production was also significantly positively 506 correlated with metacaspase activity at all days (Fig. 4c, p=0.03) further indicating that biomass 507 undergoing PCD produced more TEP. In the diatom Rhizosolenia setigera TEP concentrations 508 increased during the stationary- decline phase (Fukao et al., 2010) and could also affect buoyancy. 509 Coupling between PCD and elevated production of TEP and aggregation has been previously shown in 510 Trichodesmium cultures (Berman-Frank et al., 2007;Bar-Zeev et al., 2013). Here we cannot confirm a 511 mechanistic link between nutrient stress, PCD induction, and TEP production, but show significant 512 correlations between these parameters measured at LDA and LDB with the declining diazotroph 513 blooms (Fig. 4c) (Spungin et al., 2016).

514 Furthermore, TEP concentrations at LDB were significantly positively correlated with TOC, 515 POC, and DOC (Fig. 4d-f) confirming the integral part of TEP in the cycling of carbon at this station. 516 Assuming a carbon content of 63 % (w/w), (Engel, 2004) we estimate that TEP contributes to the 517 organic carbon pool in the order of ~ 80-400 µg C L-1 (Table 1 and Table 2) with the percentage of 518 TEP-C from TOC ranging between 0.08- 42 % and 11-32 % at LDA and LDB respectively (Table 1 519 and 2, taking into account spatial and temporal differences). Thus, at LDB, surface TEP-C increased 520 from 22 % at day 3 to 32 % of the TOC content at day 5. Yet, for the same time period a 2-fold

521 increase of TEP was measured at 200 m (11 % to 21 %). These results reflect the bloom status at LDB. 522 During bloom development; organic C and N are incorporated to the cells and little biotic TEP 523 production occurs while stationary growth (as long as photosynthesis continues) stimulates TEP 524 production (Berman-Frank and Dubinsky, 1999). When mortality exceeds growth, the presence of 525 large amounts of sticky TEP provide “hot spots” or substrates for bacterial activity and facilitate 526 aggregation of particles and enhanced sinking rates of aggregates as previously observed for 527 Trichodesmium (Bar-Zeev et al., 2013).

528 3.4. Linking PCD-induced bloom demise to particulate C and N export

529 Measurements of elevated rates of metacaspase and caspase-like activities and changes in TEP 530 concentrations are not sufficient to link PCD and vertical export of organic matter as demonstrated for 531 laboratory cultures of Trichodesmium (Bar-Zeev et al., 2013). To see whether PCD-induced mortality 532 led to enhanced carbon flux at sea we now examined mass flux and specific evidence for diazotrophic 533 contributions from the drifting sediment traps (150, 325 and 500 m) at LDA and LDB stations.

534 Mass flux at LDA increased with time, with the maximal mass flux rates obtained from the 150 m 535 trap (123 dry weight (DW) m-2 d-1) on day 4. The highest mass flux was 40 and 27 DW m-2 d-1 from 536 the deeper sediment traps (325 and 500 m traps respectively). Particulate C (PC) and particulate 537 nitrogen (PN) showed similar trends as the mass flux. At LDA, PC varied between 3.2-30 mg sample-1 538 and PN ranged from 0.3-3.2 mg sample-1 in the 150 m trap. At LDB, PC varied from 1.6 to 6 mg 539 sample-1 and total PN ranged from 0.2 to 0.8 mg sample-1 in the 150 m trap. The total sediment flux in 540 the traps deployed at LDB ranged between 6.4 mg m-2 d-1 (150 m, day 4) and 33.5 mg m-2 d-1 (500 m, 541 day 2), with an average of 18.9 mg m-2 d-1. Excluding the deepest trap at 500 m where the high flux 542 occurred at day 2, in the other traps the highest export flux rate occurred at the last day at the station 543 (day 5).

544 Analyses of the community found in the sediment traps, determined by qPCR from the 545 accumulated matter on day 5 at the station, confirmed that Trichodesmium, UCYN-B and het-1 were 546 the most abundant diazotrophs in the sediment traps at LDA and LDB stations (Caffin et al., 2017), 547 significantly correlating with the dominant diazotrophs found at the surface of the ocean (measured on 548 day 1). Trichodesmium and Rhizosolenia-Richelia association (het-1) were the major contributors to 549 diazotroph export at LDA and LDB while UCYN-B and het-1 were the major contributors at LDC 550 (Caffin et al., 2017). At LDA the deeper traps contained Trichodesmium with 2.6 x107 and 1.4x107 551 nifH copies L-1 at the 325 and 500 m traps respectively. UCYN-B was detected in all traps with the 552 highest abundance at the 325 m (4.2x106 nifH copies L-1) and 500 m traps (2.8x106 nifH copies L-1). 553 Het-1 was found only in the 325 m trap with 2.0x107 nifH copies L-1 (Fig. 5a). At LDB, 554 Trichodesmium, UCYN-B and het-1 were detected at the 325 and 500 m traps but not at 150 m. 555 Trichodesmium counts were 9x105 at the 325 m trap and 5x106 nifH copies L-1 for the 500 m trap (Fig.

556 5b). While evidence for UCYN-B showed 3.6x105 and 10x105 nifH copies L-1 at 325 and the 500 m 557 traps respectively (Fig. 5b).

558 In addition to exported Trichodesmium and Rhizosolenia-Richelia association, the small 559 unicellular UCYN-B (< 4 µm) were also found in the sediment traps, including the deeper (500 m) 560 traps. UCYN-B is often associated with larger phytoplankton such as the diatom Climacodium 561 frauenfeldianum (Bench et al., 2013) or in colonial phenotypes (> 10 µm fraction) as has been 562 observed in the northern tropical Pacific (ALOHA) (Foster et al., 2013). Sedimenting UCYN-B were 563 detected during the VAHINE mesocosm experiment in the New Caledonian lagoon in shallow (15m) 564 sediment traps) (Bonnet et al., 2015) and were also highly abundant in a floating sediment trap 565 deployed at 75 m for 24 h in the North Pacific Subtropical Gyre (Sohm et al., 2011). Thus our data 566 substantiates earlier conclusions that UCYN, which form large aggregates (increasing actual size and 567 sinking velocities), can efficiently contribute to export in oligotrophic systems (Bonnet et al., 2015). 568 Increase in aggregate size could also occur with depth, possibly due to the high concentrations of TEP 569 produced at the surface layer, sinking in the water column, providing a nutrient source and enhancing 570 aggregation (Berman-Frank et al., 2016).

571 The sinking rates of aggregates in the water column, depend on factors such as fluid viscosity, 572 particle source material, morphology, density, and variable particle characteristics. Sinking velocities 573 of diatoms embedded in aggregates are generally fast (50-200 m d-1) (Asper, 1987;Alldredge, 1998) 574 compared with those of individually sinking cells (1-10 m d-1) (Culver and Smith, 1989) allowing 575 aggregated particles to sink out of the photic zone to depth. Assuming a sinking rate of 576 Trichodesmium-based aggregates of 150-200 m d-1 (Bar-Zeev et al., 2013), we would need to shift the 577 time frame by 1 day to see whether PCD measured from the surface waters is coupled with changes in 578 organic matter reflected in the 150 m sediment traps. Thus, at LDA, examining metacaspase activities 579 from the surface with mass flux and particulate matter obtained 24 h later yielded a significant positive 580 correlation between these two parameters (Fig. 5c).

581 LDA had the highest export flux and particulate matter found in its traps relative to LDB and 582 LDC. Diazotrophs contributed ~ 36 % to PC export in the 325 m trap at LDA, with Trichodesmium 583 comprising the bulk of diazotrophs (Caffin et al., 2017) In contrast, at LDB, we found lower flux rates 584 and lower organic material in the traps with Trichodesmium contributing the bulk of diazotroph 585 biomass at the 150 m trap. We believe that at LDB the decline phase began only halfway through our 586 sampling and thus the resulting export efficiency we obtained for the 5 days at station was relatively 587 low compared to the total amount of surface biomass. Moreover, considering export rates, and the 588 experimental time frame, most of the diazotrophic population may have been directly exported to the 589 traps only after we left the station (i.e. time frame > 5 days). This situation is different from the bloom 590 at LDA, where enhanced mortality, biomass deterioration, and bloom crash were initiated 1-2 weeks

591 before our arrival and sampling at the station. Thus, at LDA, elevated mass flux and higher 592 concentrations of organic matter were obtained from all three depths of the deployed traps.

593 In the field, especially in the surface layers of the oligotrophic oceanic regions, dead cells are rarely 594 seen at later stages (Berges and Choi 2014) or not seen (Segovia et al., 2018). This is due to the fact 595 that dying and dead cells are utilized quickly and recycled within the food web and upper surface layer. 596 However, under bloom conditions, when biomass is high, the fate of the extensive biomass is more 597 complicated (Bonnet et al., 2015). PCD induced cell death, combined with buoyancy loss, can lead to 598 rapid sinking to depth of the biomass at a speed that would prevent large feeding events on this biomass. 599 This may be determined by POC downward fluxes easy to measure in the lab and extremely complex 600 in the open ocean. We previously measured POC export in our lab under controlled conditions (Bar- 601 Zeev et al., 2013). Here, using sediment traps we have measured POC fluxes, but also have specific 602 indications (NifH reads) of Trichodesmium and other diazotrophs which were blooming for several days 603 at the surface. This indicates that under bloom conditions when biomass is high some of the cell pellets 604 do sink down out of the food web. 605

606 4. Conclusion and implications 607 Our specific objective in this study was to examine whether diazotroph mortality mediated by 608 PCD can lead to higher fluxes of organic matter sinking to depth. The OUTPACE cruise provided this 609 opportunity in two out of three long-duration (5 day) stations where large surface blooms of 610 diazotrophs principally comprised of Trichodesmium, UCYN-B and diatom-diazotroph associations 611 Rhizosolenia-Richelia and were encountered. We demonstrate (to our knowledge for the first time) 612 metabolically active metacaspases in oceanic populations of Richelia and Trichodesmium. Moreover, 613 metacaspase activities were significantly correlated to caspase-like activities at both LDA and LDB 614 stations with both proteins families characteristic of PCD induced mortality. Evidence from drifting 615 sediment traps, deployed for 5 days at the two stations, showed high TEP concentrations formed at 616 surface and shifting to depth, increasing numbers of diazotrophs in sediment traps from 150, 350, 500 617 m depths), and a time-shifted correlation between metacaspase activity (signifying PCD) and vertical 618 flux of PC and PN). 619 Yet, our results also delineate the natural variability of biological oceanic populations. The two 620 stations, LDA and LDB were characterized by biomass at physiologically different stages. The 621 biomass from LDA displayed more pronounced mortality that had begun prior to our arrival at station. 622 In contrast, satellite data indicated that at LDB, the surface Trichodesmium bloom was sustained for at 623 least a month prior to the ship’s arrival and remained high for the first 3 days of our sampling before 624 declining by 40 % at day 5. As sediment trap material was examined during a short time frame, of 625 only 5 days at each LD station, we assume that a proportion of the sinking diazotrophs and organic 626 matter were not yet collected in the traps and had either sunk before trap deployment or would sink

627 after we left the stations. Thus, these different historical conditions which influence physiological 628 status at each location also impacted the specific results we obtained and emphasized a-priori the 629 importance of comprehensive spatial and temporal sampling that would facilitate a more holistic 630 understanding of the dynamics and consequences of bloom formation and fate in the oceans. 631 632 Author contributions

633 IBF, DS, and SB conceived and designed the investigation linking PCD to vertical flux within the 634 OUTPACE project. NB, MS, AC, MPP, NL CD and RAF participated, collected and performed analyses 635 of samples, DS analysed samples and data. DS and IBF wrote the manuscript with contributions from 636 all co-authors.

637 638 Acknowledgments 639 This research is a contribution of the OUTPACE (Oligotrophy from Ultra-oligoTrophy PACific 640 Experiment) project (https://outpace.mio.univ-amu.fr/) funded by the Agence Nationale de la 641 Recherche (grant ANR-14-CE01-0007-01), the LEFE-CyBER program (CNRS-INSU), the Institut de 642 Recherche pour le Développement (IRD), the GOPS program (IRD) and the CNES (BC T23, ZBC 643 4500048836). The OUTPACE cruise (http://dx.doi.org/10.17600/15000900) was managed by the MIO 644 (OSU Institut Pytheas, AMU) from Marseilles (France). The authors thank the crew of the R/V 645 L'Atalante for outstanding shipboard operations. G. Rougier and M. Picheral are warmly thanked for 646 their efficient help in CTD rosette management and data processing, as well as C. Schmechtig for the 647 LEFE-CyBER database management. Aurelia Lozingot is acknowledged for the administrative work. 648 All data and metadata are available at the following web address: http://www.obs- 649 vlfr.fr/proof/php/outpace/outpace.php. We thank Olivier Grosso (MIO) and Sandra Hélias (MIO) for 650 the phosphate data and François Catlotti (MIO) for the zooplankton data. The ocean color satellite 651 products were provided by CLS in the framework of the CNES-OUTPACE project (PI A.M. Doglioli) 652 and the video is courtesy of A. de Verneil. RAF acknowledges Stina Höglund and the Image Facility 653 of Stockholm University and the Wenner-Gren Institute for access and assistance in confocal 654 microscopy. The participation of NB, DS, and IBF in the OUTPACE experiment was supported 655 through a collaborative grant to IBF and SB from Israel Ministry of Science and Technology Israel 656 and the High Council for Science and Technology (HCST)-France 2012/3-9246, and United States- 657 Israel Binational Science Foundation (BSF) grant No. 2008048 to IBF. RAF was funded by the Knut 658 and Alice Wallenberg Stiftelse, and acknowledges the helpful assistance of Dr. Lotta Berntzon. This 659 work is in partial fulfillment of the requirements for a PhD thesis for D. Spungin at Bar-Ilan 660 University.

661

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857 Figure legends 858 859 Figure 1- Temporal dynamics of surface chlorophyll-a concentrations in the long duration (LD) 860 stations (a) LDA (b) LDB and (c) LDC station. Chlorophyll a was measured over 5 days at each 861 station (marked in gray). Satellite data of daily surface chlorophyll a (mg m-3) around the long duration 862 stations of OUTPACE was used to predict changes in photosynthetic biomass before and after our 863 measurements at the station (marked as dashed lines). Satellite data movies are added as 864 supplementary data (Supplementary videos S1, S2, S3). Chlorophyll a profiles in (d) LDA (e) LDB 865 and (f) LDC. Measurements of Chl a were taken on days 1 (black dot), 3 (white triangle) and 5 (grey 866 square) at the LDB station at 5 depths between surface and 200 m depths. 867 868 Figure 2- (a-d) Microscopic images of Trichodesmium from LDA and LDB. Observations of poor cell 869 integrity were reported for collected samples, with filaments at various stages of degradation and 870 colony under possible stress. (e) Confocal and (d) processed IMARIS images of Rhizosolenia-Richelia 871 symbioses (het-1) at 6m (75 % surface incidence). Green fluorescence indicates the chloroplast of the 872 diatoms, and red fluorescence are the Richelia filaments; Microscopic observations indicate that near 873 the surface Rhizosolenia populations were already showing signs of decay since the silicified cell-wall 874 frustules were broken and free filaments of Richelia were observed. Images by Andrea Caputo. 875 876 Figure 3- PCD indices from LDA and LDB (a) Caspase-like activity from LDA (pM hydrolyzed mg 877 protein-1 min-1) assessed by cleavage of the canonical fluorogenic substrate, z-IETD-AFC. (b) 878 Metacaspase activity from LDA (pM hydrolyzed mg protein-1 min-1) assessed by cleavage of the 879 canonical fluorogenic substrate, Ac-VRPR-AMC. (c) Relationship between caspase-like activity and 880 metacaspase activity from LDA (r=0.7, n=15, p=0.005). (d) Caspase-like activity rats in LDB station 881 (pM hydrolyzed mg protein-1 min-1), (e) Metacaspase activity in LDB station (pmol hydrolyzed mg 882 protein-1 min-1), (f) Relationship between caspase-like activity and metacaspase activity in LDB station 883 (r=0.7, n=15, p=0.001). Caspase-like and metacaspase activates at LDA and LDB stations were 884 measured on days: 1(black dot), 3 (white triangle) and 5 (grey square) between surface and 200 m. 885 Error bars represent ± 1 standard deviation (n=3). 886 887 Figure 4- (a) Depth profiles of TEP concentrations (µg GX L-1) at LDB station. Measurements were 888 taken on days 1, 3 and 5 at the station at surface-200 m depths. (b) The relationships between the 889 concentration of transparent exopolymeric particles (TEP), (µg GX L-1) and dissolved inorganic 890 phosphorus DIP (µmol L-1) for days 1, 3 and 5 at the LDB station (r=-0.7, n=15, p=0.005). 891 Relationships between the concentration of transparent exopolymeric particles (TEP), (µg GX L-1) and 892 (c) metacaspase activity (pmol hydrolyzed mg protein-1 min-1) for days 1, 3 and 5 at the LDB assessed 893 by cleavage of the canonical fluorogenic substrate, Ac-VRPR-AMC (r =0.6 n=15, p=0.03); (d) and

894 with dissolved organic carbon (DOC), (µM) for days 1, 3 and 5 at the LDB station (r=0.7, n=15, 895 p=0.004) (e) and with particulate organic carbon (POC) (µM) for days 1, 3 and 5 at the LDB station 896 (r=0.8, n=5, p=0.1 for day 1 and r=0.9, n=8 p=0.002 for day 3and 5) (f) and with total organic carbon 897 (TOC) (µM) for days 1, 3 and 5 at the LDB station (r=0.7, n=15, p=0.001). Measurements were taken 898 on days 1 (black dot), 3 (white triangle) and 5 (grey square) at LDB at 5 depths between surface and 899 200 m depths. Error bars for TEP represent ± 1 standard deviation (n=3). 900 901 Figure 5- (a) Diazotrophic abundance (nifH copies L-1) of Trichodesmium (dark grey bars); UCYN-B 902 (white bars); and het-1 (light grey bars) recovered in sediment traps at the long duration stations (A) 903 Diazotrophic abundance (nifH copies L-1) observed in the traps at LDA station (b) Diazotrophic 904 abundance (nifH copies L-1) observed in the traps at LDB station. Abundance was measured from the 905 accumulated material on day 5 at each station. Sediment traps were deployed at the LD station at 150 906 m, 325 m, and 500 m. Error bars represent ± 1 standard deviation (n=3). (c) Relationship between 907 metacaspase activity (pmol hydrolyzed mg protein-1 min-1) measured at the surface waters of LDA 908 station assessed by cleavage of the canonical fluorogenic substrate, Ac-VRPR-AMC and mass flux 909 rates (mg m2 h-1) (green circle), particulate carbon (PC, mg sample-1) (green triangle) and particulate 910 nitrogen (PN, mg sample-1) (green square) measured in the sediment trap deployed at 150 m. A 1-day 911 shift between metacaspase activities at the surface showed a significant positive correlation with mass 912 flux and particulate matter obtained in the sediment trap at LDA station at 150 m. 913 914 915 916 917 918 919 920 921 922 923 924 925 926

927 Table 1- Temporal changes in the relative composition (w/w) and distribution of TEP, TEP-C and 928 organic carbon and nitrogen fractions within the water column during days 1,3 and 5 in the LDA 929 station at different depth ranging between surface (10 m) to 200 m. 930 Day at Depth TEP TEP-C %TEP-C POC TOC POC/PON LDA (m) (µg GX L-1) (µM) (µM) station 1 200 296±135 186.5 27.2 3.04 57.2 5 150 ND ND ND 3.18 61.1 13 70 87±17 54.8 6.7 2.93 68.7 11 50 562±7 354.3 41.9 2.47 70.5 13 10 241±40 152.3 14.5 9.21 87.4 8 3 200 191±13 120.9 18.6 1.29 54.2 27 150 144±54 91.2 12.9 2.22 59.0 22 80 263 166.1 20.5 4.62 67.5 15 10 126±2 79.6 8.3 3.60 79.7 12 5 200 200 126 21.3 2.84 54.2 236 150 220 138.6 18.0 2.72 58.2 7 80 146 92.2 12.1 4.91 63.3 8 50 348±60 219.5 26.8 3.33 68.3 6 10 ND ND ND 5.80 83.7 7 931 932 933 934 Table 2- Temporal changes in the relative composition (w/w) and distribution of TEP, TEP-C and 935 organic carbon and nitrogen fractions within the water column during days 1,3 and 5 in the LDB 936 station at different depth ranging between surface (7 m) to 200 m. 937 Day at Depth TEP TEP-C %TEP-C POC TOC POC/PON LDB (m) (µg GX L-1) (µM) (µM) station 1 7 408±36 257.1 23.4 8.95 91.5 6.0 35 279±86 175.9 17.0 5.86 86.0 9.1 100 214±67 134.7 16.8 ND 66.7 ND 150 145±34 91.5 12.3 3.79 61.9 11.2 200 244±113 153.7 20.3 7.61 63.2 9.8 3 7 402±12 253.1 22.5 8.88 93.9 6.9 35 193±48 121.8 12.6 3.07 80.3 8.2 100 163±33 102.4 12.6 ND 67.8 ND 150 145±34 91.6 12.0 1.91 63.8 7.4 200 127±79 80.2 11.3 1.71 59.3 5.7 5 7 565±87 355.8 32.5 5.32 91.3 5.9 70 294±53 185.2 20.1 2.21 76.7 6.1 100 264±160 166.2 19.6 2.25 70.6 8.0 150 224±51 140.8 15.9 1.53 73.9 5.1 200 231±45 145.8 21.1 1.11 57.6 5.5 938 939 Abbreviations: TEP, transparent exopolymeric particle; TEP-C, TEP carbon; POC, particulate organic 940 C; TOC, total organic C; ND- no data. 941

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